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- PDB-9of2: Dimer of HIF-2a-ARNT Heterodimers Complexed on 51-bp HRE/HAS -

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Basic information

Entry
Database: PDB / ID: 9of2
TitleDimer of HIF-2a-ARNT Heterodimers Complexed on 51-bp HRE/HAS
Components
  • 51-nt Hypoxia Response Element (Forward)
  • 51-nt Hypoxia Response Element (Reverse)
  • Aryl hydrocarbon receptor nuclear translocator
  • Endothelial PAS domain-containing protein 1
KeywordsDNA BINDING PROTEIN / Complex / Hypoxia / Transcription / Cancer / Dimer / Higher-ordered
Function / homology
Function and homology information


myoblast fate commitment / nuclear aryl hydrocarbon receptor complex / Aryl hydrocarbon receptor signalling / positive regulation of hormone biosynthetic process / aryl hydrocarbon receptor complex / Cellular response to hypoxia / Transcriptional regulation of pluripotent stem cells / regulation of protein neddylation / PTK6 Expression / positive regulation of protein sumoylation ...myoblast fate commitment / nuclear aryl hydrocarbon receptor complex / Aryl hydrocarbon receptor signalling / positive regulation of hormone biosynthetic process / aryl hydrocarbon receptor complex / Cellular response to hypoxia / Transcriptional regulation of pluripotent stem cells / regulation of protein neddylation / PTK6 Expression / positive regulation of protein sumoylation / norepinephrine metabolic process / Xenobiotics / surfactant homeostasis / Phase I - Functionalization of compounds / positive regulation of vascular endothelial growth factor receptor signaling pathway / epithelial cell maturation / Regulation of gene expression by Hypoxia-inducible Factor / aryl hydrocarbon receptor binding / blood vessel remodeling / positive regulation of vascular endothelial growth factor production / Endogenous sterols / embryonic placenta development / cis-regulatory region sequence-specific DNA binding / positive regulation of endothelial cell proliferation / NPAS4 regulates expression of target genes / visual perception / regulation of heart rate / Pexophagy / erythrocyte differentiation / positive regulation of erythrocyte differentiation / positive regulation of glycolytic process / RNA polymerase II transcription regulatory region sequence-specific DNA binding / mitochondrion organization / lung development / mRNA transcription by RNA polymerase II / transcription coactivator binding / PPARA activates gene expression / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / multicellular organismal-level iron ion homeostasis / negative regulation of inflammatory response / RNA polymerase II transcription regulator complex / nuclear receptor activity / sequence-specific double-stranded DNA binding / Neddylation / positive regulation of cold-induced thermogenesis / DNA-binding transcription activator activity, RNA polymerase II-specific / cellular response to oxidative stress / angiogenesis / cellular response to hypoxia / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / response to oxidative stress / sequence-specific DNA binding / cell differentiation / DNA-binding transcription factor activity, RNA polymerase II-specific / response to hypoxia / nuclear speck / nuclear body / RNA polymerase II cis-regulatory region sequence-specific DNA binding / protein heterodimerization activity / DNA-binding transcription factor activity / regulation of transcription by RNA polymerase II / chromatin / signal transduction / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
HIF-1 alpha, transactivation domain, C-terminal / HIF-1 alpha C terminal transactivation domain / : / Hypoxia-inducible factor 1-alpha bHLH domain / Hypoxia-inducible factor, alpha subunit-like / Hypoxia-inducible factor-1 / Nuclear translocator / Helix-loop-helix DNA-binding domain / PAC motif / Motif C-terminal to PAS motifs (likely to contribute to PAS structural domain) ...HIF-1 alpha, transactivation domain, C-terminal / HIF-1 alpha C terminal transactivation domain / : / Hypoxia-inducible factor 1-alpha bHLH domain / Hypoxia-inducible factor, alpha subunit-like / Hypoxia-inducible factor-1 / Nuclear translocator / Helix-loop-helix DNA-binding domain / PAC motif / Motif C-terminal to PAS motifs (likely to contribute to PAS structural domain) / PAS domain / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. / Helix-loop-helix DNA-binding domain superfamily / PAS fold / PAS fold / PAS domain / PAS repeat profile. / PAS domain / PAS domain superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / Aryl hydrocarbon receptor nuclear translocator / Endothelial PAS domain-containing protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsClosson, J.D. / Xu, X. / Gardner, K.H.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)U54 CA132378 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)U54 C137788 United States
The G. Harold and Leila Y. Mathers FoundationMF-2112-02288 United States
CitationJournal: bioRxiv / Year: 2025
Title: CONTEXT-DEPENDENT VARIABILITY OF HIF HETERODIMERS INFLUENCES INTERACTIONS WITH MACROMOLECULAR AND SMALL MOLECULE PARTNERS.
Authors: Joseph D Closson / Xingjian Xu / Meiling Zhang / Tarsisius T Tiyani / Leandro Pimentel Marcelino / Eta A Isiorho / Jason S Nagati / Joseph A Garcia / Kevin H Gardner /
Abstract: Hypoxia inducible factors (HIFs) are transcription factors that coordinate cellular responses to low oxygen levels, functioning as an α/β heterodimer which binds a short hypoxia response element ...Hypoxia inducible factors (HIFs) are transcription factors that coordinate cellular responses to low oxygen levels, functioning as an α/β heterodimer which binds a short hypoxia response element (HRE) DNA sequence. Prior studies suggest HIF/HRE complexes are augmented by the binding of additional factors nearby, but those interactions are not well understood. Here, we integrated structural and biochemical approaches to investigate several functionally relevant HIF assemblies with other protein, small molecule, and DNA partners. First, we used cryo-electron microscopy (cryo-EM) to establish HIF-1 and HIF-2 form novel "dimer-of-heterodimers" (DoHD) complexes on extended human EPO enhancer sequences, showing that one heterodimer bound at a canonical HRE site with the second binding in an inverted fashion to an HRE-adjacent sequence (HAS) 8 bp away. Consistent with ARNT PAS-B domains predominating interactions within a DoHD, we found HIF-1 and HIF-2 assemble mixed DoHD complexes on the same DNA. Second, we saw substantial variability among ligands for isolated ARNT or HIF-2α PAS-B domains to bind larger complexes: for example, the ARNT PAS-B binding KG-548 and KG-279 ligands both bound the simpler HIF-2 heterodimer but exhibited differential binding to a HIF-2 DoHD. Finally, we combined cryo-EM and hydrogen-deuterium exchange by mass spectrometry (HDX-MS) to show how HIF-1 and HIF-2 heterodimers engage the transforming acidic coiled-coil containing protein 3 (TACC3) coactivator via both ARNT and HIF-α subunits, though this was unseen in the larger DoHD. Our findings highlight the importance of both molecular context and dynamics in biomolecular complex formation, adding to the complexities of potential regulation.
History
DepositionApr 29, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 11, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 25, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 51-nt Hypoxia Response Element (Forward)
B: 51-nt Hypoxia Response Element (Reverse)
C: Endothelial PAS domain-containing protein 1
D: Aryl hydrocarbon receptor nuclear translocator
E: Endothelial PAS domain-containing protein 1
F: Aryl hydrocarbon receptor nuclear translocator


Theoretical massNumber of molelcules
Total (without water)202,9246
Polymers202,9246
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: DNA chain 51-nt Hypoxia Response Element (Forward)


Mass: 15822.080 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: GenBank: 182197
#2: DNA chain 51-nt Hypoxia Response Element (Reverse)


Mass: 15608.979 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: GenBank: 182197
#3: Protein Endothelial PAS domain-containing protein 1 / EPAS-1 / Basic-helix-loop-helix-PAS protein MOP2 / Class E basic helix-loop-helix protein 73 / ...EPAS-1 / Basic-helix-loop-helix-PAS protein MOP2 / Class E basic helix-loop-helix protein 73 / bHLHe73 / HIF-1-alpha-like factor / HLF / Hypoxia-inducible factor 2-alpha / HIF-2-alpha / HIF2-alpha / Member of PAS protein 2 / PAS domain-containing protein 2


Mass: 42684.453 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal 21 residues (MGSSHHHHHHSQDPGKEKKRS) are vector-derived
Source: (gene. exp.) Homo sapiens (human) / Gene: EPAS1, BHLHE73, HIF2A, MOP2, PASD2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q99814
#4: Protein Aryl hydrocarbon receptor nuclear translocator / ARNT protein / Class E basic helix-loop-helix protein 2 / bHLHe2 / Dioxin receptor / nuclear ...ARNT protein / Class E basic helix-loop-helix protein 2 / bHLHe2 / Dioxin receptor / nuclear translocator / Hypoxia-inducible factor 1-beta / HIF-1-beta / HIF1-beta


Mass: 43061.820 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARNT, BHLHE2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P27540
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dimer of two HIF-2a-ARNT heterodimers complexed on 51-bp HRE
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.202 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4 / Details: 50mM HEPES, 150mM NaCl, 5mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidC8H18N2O4S1
2150 mMSodium ChlorideNaCl1
35 mMMagnesium ChlorideMgCl21
SpecimenConc.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 4000 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm
Image recordingElectron dose: 58.94 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 10910

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Processing

EM software
IDNameVersionCategory
1Warp1.0.9particle selection
2Leginon3.7image acquisition
4Warp1.0.9CTF correction
7UCSF ChimeraX1.9model fitting
9PHENIX1.20.1_4487model refinement
10Coot0.9.6.1.1model refinement
13cryoSPARC4.7.0classification
14cryoSPARC4.7.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 656778
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 164352 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0041240
ELECTRON MICROSCOPYf_angle_d0.5941909
ELECTRON MICROSCOPYf_dihedral_angle_d37.216534
ELECTRON MICROSCOPYf_chiral_restr0.03216
ELECTRON MICROSCOPYf_plane_restr0.00354

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